Enantiomer Separation of Chiral Acids on CHIRALPAK...Polar Organic Conditions Preferred due to...

Enantiomer Separation of Chiral Acids on

CHIRALPAK® AX-QN and

CHIRALPAK® AX QD

face-to-face

p -stacking amide-type

hydrogen bond

charge-supported

hydrogen bond

van der Waals

interaction

Cl-tBuCQN

DNB-(S)-Leu

N.M. Maier et al. Chirality (1999)

N.M. Maier et al. JACS (2001)

Non-covalent Interactions Stabilizing the Quinine

tert-butylcarbamate / 3,5-dinitrobenzoyl leucine complex (X-Ray Crystal Structure)

Chiral Recognition Mechanism

Principles of Analyte Retention on

CHIRALPAK® AX Columns:

Weak Anion Exchange

SAdissSOprottotX

SOKk ,,][

1][

k retention factor

K association constant

phase ratio

[SO]tot total selector concentration

[X-] counterion concentration

prot,SO degree of protonation of selector

diss, SA degree of dissociation of analyte

pH

R3NH+

R3N

pKs,SO

%SOprot

1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

25

50

75

100

R-COO-

R-COOH

%SAdiss

pKs,SA

0

25

50

75

100

Impact of pH on Retention

Enantioselectivity

2.5

2.0

1.5

1.0

0.5

0.0

Retention and enantioselectivity can be optimized by appropriate

adjustment of the pHa of the mobile phase.

M. Lämmerhofer et al., Am.Lab. (2002)

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5

pHa

k1

k2

CHIRALPAK AX-QN (150 x 4 mm I.D.); MeOH-aqu. 0.2 M NH4OAc (80:20, v/v);

1.0 mL/min; 254 nm; T 25 °C

Analyte: N-benzoyl leucine

Impact of Mobile Phase pH on

Retention & Enantioselectivity

Retention adjustable via counterion concentration without significant

loss in enantioselectivity.

M. Lämmerhofer et al., Am.Lab. (2002)

log [CH3COO-]

-0.2 -0.6 -1.0 -1.4 -1.8

0.8

0.6

0.4

0.2

0.0

-0.2

-0.4

-0.6

log

log k2

log k1

log

k, lo

g

Impact of Counterion

Concentration on Retention &

Enantioselectivity

CHIRALPAK AX-QN (150 x 4 mm I.D.); MeOH-aqu. 0.2 M NH4OAc (80:20, v/v);

1.0 mL/min; 254 nm; T 25 °C

Analyte: N-benzoyl leucine

Method Development Strategy:

Optimization Parameter

pH (pH profile of k and alpha)

Counterions (nature, concentration)

Organic modifier (nature, relative amounts)

Lower flow rates: Strongly electrostatic SO-SA interaction – slow

mass transfer.

Low temperature: Chiral recognition process enthalpically driven

– low temperature favor enantioselectivity.

Polar Organic Conditions

Preferred due to enhanced enantioselectivity, generality, low

viscosity, solubility of most analyte

Little non-specific retention of lipophilic compounds

MS-compatible

Methanol - ammonium acetate - acetic acid (98:2:0.5 to 95:5:2)

Methanol - triethylamine - acetic acid

Methanol - ammonium format - formic acid

Methanol - acetic acid

Acetonitrile - acetic acid

POM works for most N-acylated amino acids, N-blocked

peptides, carboxylic acid and drug compounds with pKa 3 to 5.

M. Lämmerhofer et al., J. Pharm. Biomed. Anal. (2004)

NH

COOH

Cl

Cl

H3COOC

Polar Organic Conditions: Dihydropyrimidine Carboxylic Acid

Ca2+-Channel Blocker

Intermediate

CHIRALPAK AX-QN (150 x 4.6 mm ID), 0.124 % AcOH in acetonitrile; 1.0 mL/min; 250 nm; 25°C.

W. Bicker et al., J.Chromatogr. A (2004)

Polar Organic Conditions:

Pyrethroic Acids

COOH

cis/trans-Chrysanthemic acid

(Pesticide Intermediate)

a) Polar-organic conditions: 0.06% AcOH in acetonitrile-methanol 95:5 (v/v); 0.65 mL/min; 25°C.

b) Reversed-phase conditions: 10mM AcOH in acetonitrile-water

90:10 (v/v), pHa 6.0 (NH3 aq.), 0.65 mL/min; 25°C.

a

b

Reversed Phase Conditions

Exploits mixed reversed phase/anion exchange mechanisms

For analytes poorly soluble/retained under polar organic

conditions:

Phosphoric acids, sulfonic, phosphonic, phosphinic acids, poly-

carboxylic acids

Aqueous ammonium acetate – methanol (pHa 4.5 to 7.0)

Aqueous sodium phosphate – methanol/acetonitrile (pHa 4.5 to 7.0)

Other buffers: Citrate >> phosphate > format > acetate

Column life time may be shortened due to exposure to strong

buffers. Column may need regeneration after use of citrate.

W. Bicker et al., J.Chromatogr. A (2004)

Reversed Phase Conditions:

Phosphinic Acids

CHIRALPAK AX-QN (150 x 4.6 mm ID), methanol-50 mM sodium phosphate buffer (80:20, v/v), pHa 5.6; 1.0 mL/min; 250 nm; 40°C.

Lämmerhofer et al., Tetrahedron Asym. (2003)

Reversed Phase Conditions:

Phosphinic Acid Derivatives

CHIRALPAK AX-QN (150 x 4.6 mm ID), methanol-50 mM sodium phosphate buffer (80:20, v/v), pHa 5.6; 1.0 mL/min; 250 nm; 40°C.

H. Gika et al., J. Chromatogr. B (2004)

Reversed Phase Conditions:

Amino Acid - Thyroxine

Chiralpak®QN-AX (150 x 4.6 mm ID), ACN-0.05 NH4OAc (60:40 v/v); 0.7 mL/min; 240 nm; 25°C

I

O

I

NH2

COOHHO

I

R1

Thyroxine (T4): R1 = I

Triiodothyronine (T3): R1 = H

3-MATIDA

mAu

SCOOH

NH2

HOOC

COOHH2N

HOOC

AIDA

Chiralpak QN-AX (150 x 4 mm ID); MeOH-0.1 M NH4OAc (80: 20, v/v) pHa 5.5; 1.0 mL/min; UV 254 nm; 25°C.

0.000

0.005

0.015

0.025

8.2 18.2 28.2 38.2 48.2 (min)

-0.008

-0.004

0.000

0.004

3.2 8.2 13.2 18.2 23.2 28.2 (min)

AIDA

3-MATIDA

mAu

Potential mGluR1 Subtype

Selective Antagonist

Reversed Phase Conditions:

Amino Dicarboxylic Acids

CHIRALPAK® AX-QN and AX-QD:

Pseudoenantiomeric CSPs

CHIRALPAK AX-QN Selector Co-crystallized

with DNB-(S)-leucine

CHIRALPAK AX-QD Selector

Co-Crystallized

with DNB-(R)-leucine

X-Ray Crystal Structures

min 0 4 8 12

mAU

0

10

20

30

(R)

(S)

min 0 4 8 12

mAU

0

10

20

(S)

(R)

CHIRALPAK AX-QD

Columns (150 x 4 mm ID); 1% (v/v) AcOH in MeOH; 1 mL/min, 25°C; UV 230

nm.

= 1.5

CHIRALPAK AX-QN

Control of Elution Order by

Combined Use of CHIRALPAK ®

AX-QN and AX-QD

COOH

NHO

CH3

Prep Application

Employing volatile mobile phases to facilitate product recovery

Acetonitrile - Methanol – acetic acid (99:1 to 97:3, v/v)

Acetonitrile / Methanol – formic acid (99:1 to 98:2, v/v)

SFC: CO2 – Methanol

Added acids concentrate upon solvent evaporation – chemical

stability and stereochemical integrity of the target compounds

need to be established.

Inherently favorable saturation

capacity of ion-exchangers

Possibly displacement effects

(high affinity enantiomer

sharpens front of low affinity

enantiomer

Chiralpak AX-QN (150 x 4.6 mm I.D.); 10 mM ammonium acetate + 30 mM acetic acid in methanol (pHa = 6.0); flow rate: 1 mL/min; UV 254 nm, 320 nm; 25°C.

20 µg injected

(R)

(S)

20 mg injected

= 2.1

0 5 10 15 20 (min)

0 5 10 15 20 (min)

Semiprep Separation of

N-benzoyl tert.-leucine

COOH

HN O

SFC Separation of

Diastereomeric Mixture of an

Acidic Drug Candidate

S. Staskiewicz et al., J. Label Compd Radiopharm (2007)

MeOH / CO2, no additional acidic additive

COOHR

R

CHIRALPAK® AX-QN & CHIRALPAK ®

AX-QD

Enantioselectivity for a broad range of chiral acidic compounds

Compatibility with all common HPLC solvents and modes

(non-polar, polar-organic, reversed-phase and SFC)

Stable in pH range 2 to 8

Control of elution order via pseudoenantiomeric chiral recognition

properties

Straightforward method development

Suitable for bio-analytical and MS applications

Manufacture and technical support by DAICEL

Thanks

Michael Lämmerhofer and Wolfgang Lindner

University of Vienna, Austria

All former coworkers at the Department of Analytical Chemsitry,

University of Vienna, Austria

Pilar Franco and Tong Zhang

Chiral Technologies Europe, France